EVALUATION OF ANTI-OXIDANT AND ANTI-TUMOUR ACTIVITIES OF CROTON LAEVIGATUS Vahl.
Isolation and characterization of nuclear microsatellite primers for the Barbary thuja, Tetraclinis...
Transcript of Isolation and characterization of nuclear microsatellite primers for the Barbary thuja, Tetraclinis...
MICROSATELLITE LETTERS
Isolation and characterization of nuclear microsatellite primersfor the Barbary thuja, Tetraclinis articulata (Vahl) Mast.(Cupressaceae)
Marıa Teresa Lorenzo • Ramon Casimiro-Soriguer •
Francisco Balao • Juan Luis Garcıa-Castano •
Jose M. Sanchez-Robles • Anass Terrab
Received: 23 September 2013 / Accepted: 26 September 2013 / Published online: 5 October 2013
� Springer Science+Business Media Dordrecht 2013
Abstract Fifty nuclear microsatellite primers were ini-
tially developed using next-generation sequencing (454)
data from a single individual of Tetraclinis articulata
(Vahl) Mast. Eleven primers were finally applied in 30
individuals from 3 localities from Morocco, Algeria and
Spain. The number of alleles per locus ranged from one to
ten. The average observed and expected heterozygosities
across the populations studied ranged from 0.10 to 0.80 and
from 0.09 to 0.88, respectively. The microsatellite markers
described here are valuable tools for the population genetic
research of T. articulata, and can be used to obtain infor-
mation for creating suitable management strategies to
conserve this endemic and endangered species.
Keywords Cupressaceae � Microsatellites �Population genetics � nSSR � Tetraclinis articulata
Tetraclinis Mast. is a genus of evergreen coniferous trees in
the cypress family (Cupressaceae), containing currently
only one species, the Barbary thuja. Tetraclinis articulata
(Vahl) Mast. is endemic to the mountainous regions of
North Africa (Morocco, Algeria and Tunisia), with isolated
populations occurring in Malta and S and E Spain. The
largest area of distribution is in Morocco, where it is found
in the Rif mountains (E Morocco), E & W Middle Atlas, C
& E plateau, High Atlas and Anti Atlas.
We extracted genomic DNA from leaf tissue from a
single T. articulata using an Invisorb Spin Plant Mini Kit
(Invitek, Berlin, Germany). The technique applied to
develop the microsatellites was Next Generation
Sequencing (NGS). The screening sequence data and the
design of primers were made following the procedures
described in Sanchez-Robles et al. (2012).
From 5,981 reads obtained through the NGS, only in 503
of them primers could be designed. Out of these 503 primers
pairs, we discarded 103 because of their low quality. Con-
sequently, 373 high quality primers pairs were obtained. A
total of 50 primer pairs were tested for their amplification
quality. For this, two quality DNA samples were used. Out of
the 50 loci, eleven originated clear patterns of amplification
although two of them were monomorphic (Tetra16 and
Tetra18). The rest of the selected primer pairs were discarded
as eighteen did not amplified and the other twenty-one pro-
duced unclear, difficult to score patterns.
PCR products were run on a 3,730 DNA Analyzer
sequencer (Applied Biosystem, Foster City, CA, USA) and
sized with LIZ 500 standard (Applied Biosystem, Foster
City, CA, USA). Polymerase chain reactions were per-
formed in 20 ll, the reaction mixture containing approxi-
mately 50 ng of genomic DNA, 19 PCR Buffer, 1 U/ll
i-Start Taq DNA polymerase (iNtRON Biotecnology Inc.,
Sungman, Korea), 0.25 lM primer with the 50-GTTT tail,
0.06 lM primer with the 50-CAG or the M13R tail, and
0.25 lM dye-labeled CAG or M13R universal primer with
FAM, NED or VIC fluorescent label, MgCl2, dNTP and
BSA were added in different amounts for different primer
pairs (Table 1).
M. T. Lorenzo � R. Casimiro-Soriguer � F. Balao �J. L. Garcıa-Castano � J. M. Sanchez-Robles � A. Terrab (&)
Departamento de Biologıa Vegetal y Ecologıa, Universidad de
Sevilla, Ap-1095, 41080 Sevilla, Spain
e-mail: [email protected]
M. T. Lorenzo � F. Balao
Department of Systematic and Evolutionary Botany, University
of Vienna, Rennweg 14, 1030 Vienna, Austria
R. Casimiro-Soriguer
Departamento de Biologıa, Universidad de Cadiz, Campus Rıo
San Pedro, 11510 Puerto Real, Spain
123
Conservation Genet Resour (2014) 6:233–235
DOI 10.1007/s12686-013-0064-9
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234 Conservation Genet Resour (2014) 6:233–235
123
Fragments were analyzed with the software GENE-
MARKER version 1.8 (SoftGenetics, State College, PA,
USA). We estimated the number of alleles per locus (A),
the observed and expected heterozygosity (Ho and He) and
Hardy–Weinberg equilibrium tests (HWE) for each locus
using GeneAlEx 6.5 (Peakall and Smouse 2012). Tetra44
locus showed deviation from Hardy–Weinberg equilibrium
in the Moroccan population after Bonferroni correction for
multiple comparisons (adjusted P value for 5 % nominal
level = 0.002). We calculated the null allele frequency
(An) with Micro-Checker 2.2.3 (Van Oosterhout et al.
2004) and linkage disequilibrium (LD) between pairs of
loci was tested using GENEPOP 4.0.10 software (Rousset
2008). None of the loci pairs showed significant linkage
disequilibrium (LD) in the 33 comparisons (Table 2).
We have developed eleven new nuclear microsatellite
primer pairs for T. articulata. Nine of the loci showed high
levels of polymorphism, suggesting great potential for
genetic diversity studies. These primers will enable the
development of biogeographic and conservation genetic
studies to investigate the origin of the European T. artic-
ulata populations. Additionally, the microsatellite markers
reported here provide a valuable tool for forest manage-
ment and they could be tested on other Cupressaceae
species.
Acknowledgments The authors thank L. Navarro-Sampedro for
helpful advice in the laboratory. This study was financially supported
by the Spanish Ministerio Educacion y Ciencia to AT (CGL2009-
08713). The authors thank the Herbarium and Biology Research
Services (CITIUS) of the University of Seville for allowing the use of
their facilities.
References
Don RH, Cox PT, Wainwright BJ, Baker K, Mattick JS (1991)
‘‘Touchdown’’ PCR to circumvent spurious priming during gene
amplification. Nucleic Acids Res 19:4008
Peakall R, Smouse PE (2012) GenAlEx 6.5: genetic analysis in Excel.
Population genetic software for teaching and research—an
update. Bioinformatics 28:2537–2539
Rousset F (2008) Genepop’007: a complete re-implementation of the
GENEPOP software for Windows and Linux. Mol Ecol Res
8:103–106
Sanchez-Robles JM, Balao F, Garcıa-Castano JL, Terrab A, Navarro-
Sampedro L, Talavera S (2012) Nuclear microsatellite primers
for the endangered relict fir, Abies pinsapo (Pinaceae) and cross-
amplification in related mediterranean species. Int J Mol Sci
13:14243–14250
Van Oosterhout C, Hutchinson W, Wills D, Shipley P (2004) Micro-
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errors in microsatellite data. Mol Ecol Notes 4:535–538
Table 2 Genetic diversity estimates for three Tetraclinis articulata populations
Locus Morocco (N = 10) Spain (N = 10) Algeria (N = 10)
A Ho He HWE An A Ho He HWE An A Ho He HWE An
Tetra1 3 0.300 0.395 0.667 0.068 4 0.700 0.570 0.893 0.000 6 0.600 0.550 0.993 0.000
Tetra2 7 0.700 0.800 0.278 0.056 4 0.200 0.270 0.003 0.055 8 0.700 0.805 0.137 0.058
Tetra4 1 0.000 0.000 – – 2 0.100 0.095 0.868 0.000 2 0.300 0.255 0.577 0.000
Tetra15 2 0.200 0.320 0.236 0.091 2 0.200 0.180 0.725 0.000 4 0.700 0.705 0.710 0.003
Tetra16 1 0.000 0.000 – – 1 0.000 0.000 – – 1 0.000 0.000 – –
Tetra18 1 0.000 0.000 – – 1 0.000 0.000 – – 1 0.000 0.000 – –
Tetra19 4 0.200 0.570 0.113 0.236 3 0.500 0.645 0.321 0.088 5 0.200 0.700 0.004 0.294
Tetra22 2 0.250 0.469 0.351 0.459 2 0.800 0.480 0.136 0.000 2 0.200 0.500 0.180 0.200
Tetra29 7 0.300 0.795 0.053 0.276 5 0.300 0.595 0.012 0.185 10 0.300 0.880 0.005 0.309
Tetra44 4 0.100 0.535 0.001 0.283 3 0.200 0.615 0.028 0.257 4 0.200 0.685 0.005 0.288
Tetra49 9 0.800 0.815 0.508 0.008 4 0.400 0.570 0.640 0.108 7 0.800 0.790 0.141 0.000
N sample size, A number of alleles, Ho observed heterozygosity, He expected heterozygosity, An null allele frequency, HWE Hardy–Weinberg
equilibrium test (P values)
Conservation Genet Resour (2014) 6:233–235 235
123